1. Field of the invention
The present invention relates to the assay of magnesium levels in aqueous liquids. More specifically, the present invention relates to methods for determination of serum magnesium employing .alpha.-glycerophosphate oxidase.
2. Background of the Invention
Fast and accurate determination of blood serum quantities and activity levels of various enzymes and minerals is an important medical diagnostic tool. Magnesium is the second most abundant intracellular cation present in the human body and serves as an activator for many important enzymes. Its importance in human physiology is well established. Thus, accurate assay of serum magnesium is an invaluable diagnostic tool.
Numerous spectrophotometric techniques for assaying magnesium have been developed using calmagite, methylthymol blue, titan yellow or Magon sulfonate. These methods have limited accuracy as they are based on chelation reactions and therefore are subject to varying degrees of interference by other cations present in the sample. Atomic absorption and neutron activation analysis are free from these errors, but are very complex and are prohibitively expensive for most routine clinical laboratories.
It has been found that magnesium couples with adenosine diphosphate (ADP) or adenosine triphosphate (ATP) to form a molecular complex. As reported in 31 Clinical Chemistry, pp. 703 to 705 (1985), Tabata et al have developed an enzymic magnesium assay using the enzymes hexokinase and glucose-6-phosphate dehydrogenase. In their stopped reaction assay, the amount of (NAD(P)H) produced over a 15 minute period is determined by stoppping the hexokinase reaction with tetrasodium EDTA and measuring the absorbance at 340 nm. This rate-dependent final absorbance is proportional to the amount of Mg.ATP complex present and, hence, to the serum magnesium concentration. This enzymic magnesium assay method requires a sample pre-incubation and prolonged reaction period making it unsuitable for automated instrumental analysis.
The Tabata method has a number of inherent limitations. First, it requires relatively expensive narrow-band spectrophotometers capable of measuring in the ultra-violet region to achieve the true absorbance of the coenzyme indicator. Second, the assay suffers significant interference from lipemia since the light scattering effects of this substance are tremendously enhanced at wavelengths below 400 nm. Third, assay sensitivity is limited due to the relatively low molar absorptivity of the indicator substance employed. Fourth, the assays are plagued by high reagent blanks. Fifth, the necessity to stop the reaction severely reduces the possibility of automating the procedure.
Thus, it would be desirable to develop a method which would yield the fast and accurate determination of magnesium levels in blood serum. It is also desirable to provide a method which would provide a colormetric determination with increased sensitivity. It is also desirable to provide a method which provides detectable results at a wave-length above 340 nm to minimize the light scattering effects of serum turbidity caused in lipemic samples, and places the sample in the visible rather than ultraviolet region of the spectrum.